food microbiology - the basics

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Food Microbiology – The basicsScott ColbourneBusiness Manager NSW – ALS Food & Pharmaceutical

Food Microbiology - The Basics 2

Contents

• Common Microbiological Tests - what and why

• Method Turn Around Times (TAT)

• The Confirmation Process

• Causes of counts being high initially, then low when retested


Common Tests – what and why?

• Standard Plate Count (SPC)What?A generic count of micro-organisms present. The results are non-specific Why?Gives a general indication of the level of micro-organisms present in the sample testedCan be referred to as…Total Microbial Count (TMC), Total Aerobic Microbial Count (TAMC), TVC, TVAC



• Yeast and MouldWhat?Generic test for the levels of these present. Non-specific.Why?• They cause biodegradation of natural materials, which

may become food spoilage• Some yeasts are useful in fermentation (e.g. Bread and

Beer) • However some (e.g. Candida albicans) are opportunistic

pathogens and can cause infections



• SalmonellaWhat?Pathogen found in cold/warm blooded animals and the environment

Why?• It causes food poisoning, such as gastrointestinal issues• To ensure there is no presence of pathogens



• ListeriaWhat?A genus of bacteria that contains 10 species, the most important genus, L. monocytogenes is a serious human pathogen.

Why?Healthy people and pregnant women may have mild or no symptoms, but Listeria infection may still result in miscarriage, premature birth or stillbirth.In people at risk, Listeria infection can result in serious illnesses including meningitis and septicaemia.



• E. coli and coliformsWhat?Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the faeces of warm-blooded animals and humans.

Why?• Commonly used bacterial indicators of hygiene and sanitary quality of

foods and water. • E. coli are almost exclusively of faecal origin and their presence is thus an

effective indicator of faecal contamination. • Most strains of E. coli are harmless, but some can cause serious illness in

humans.



• Staphylococcus (coagulase positive)What?A common bacterium that lives on the skin or in the nose of humans.

Why?Coagulase positive species of Staphylococcus can produce toxins in foods.



• Bacillus cereusWhat?A common bacterium found in the environment; typically associated in soil as well as a variety of foods.

Why?The toxin producing spore forming bacterium can cause vomiting and diarrhoea. Bacillus cereus spores are able to survive harsh environments which include normal cooking temperatures.


Method Turn Around Times

MethodTurnaround Time (Days)

If Clean If Confirmation is Required

Standard Plate Count 3 N/a

Yeast and Mould 5 – 7 N/a

SalmonellaELISA (Automated rapid method) 2 5

Australian Standard (manual method) 4 7

ListeriaELISA (Automated rapid method) 2 7






Standard Plate Count 3 N/a

Yeast and Mould 5 – 7 N/a

SalmonellaELISA (Automated rapid method) 2 5


ListeriaELISA (Automated rapid method) 2 7


The ELISA method (e.g. DS2) is quicker, more reliable and cheaper

The confirmation process is identical to the Australian Standard method

The next slide has more details…


Pathogens:ELISA is superior to the Australian Standard

Comparing Listeria and Salmonella test methodologiesParameter ELISA Australian Standard

Price High level of automation Labour intensive

Speed - negative 2 days 4-5 daysSpeed – confirmed positive 5-7 days 7 days

Interpretation of results Automated Labour intensiveHuman interaction Low High

NATA accreditation Yes YesInternationally recognised e.g. AFNOR Yes Yes

Client preference 95% 5%



MethodTurnaround Time TAT (Days)


Coliforms

Petrifilm 2 N/a

MPN 2 4

Presence/Absence 2 4

Plate Count 1 N/a

E.Coli

Petrifilm 2 N/a

MPN 2 6


Enterobacteriaceae

Plate Count 1 3





Coliforms

Petrifilm 2 N/a

MPN 2 4


Plate Count 1 N/a

E.Coli

Petrifilm 2 N/a

MPN 2 6


Enterobacteriaceae

Plate Count 1 3

Advantages:- Quick- Generally, no confirmation

step for coliforms

Disadvantages:- Sample matrix sensitive- Cannot use for samples with:

- High colour - High micro background- High acidity





Coliforms

Petrifilm 2 N/a

MPN 2 4


Plate Count 1 N/a

E.Coli

Petrifilm 2 N/a

MPN 2 6


Enterobacteriaceae

Plate Count 1 3

Advantages:- Quantitative- Can report to low levels (0.3)- Australian standard

Disadvantages:- Labour intensive- Statistical value – most

probable number





Coliforms

Petrifilm 2 N/a

MPN 2 4


Plate Count 1 N/a

E.Coli

Petrifilm 2 N/a

MPN 2 6


Enterobacteriaceae

Plate Count 1 3

Advantages:- Great if not detected

Disadvantages:- Qualitative only





Coliforms

Petrifilm 2 N/a

MPN 2 4


Plate Count 1 N/a

E.Coli

Petrifilm 2 N/a

MPN 2 6


Enterobacteriaceae

Plate Count 1 3

Advantages:- Quick- Quantitative

Disadvantages:- Not available for E.Coli





Coliforms

Petrifilm 2 N/a

MPN 2 4


Plate Count 1 N/a

E.Coli

Petrifilm 2 N/a

MPN 2 6


Enterobacteriaceae

Plate Count 1 3

Alternative to Coliforms as a faecal contamination indicator

Advantages:- Quick- Quantitative- Salmonella is included

Disadvantages:- Generic and positive results

may require specific testing





Staphylococcus

Plate Count 2 4-6



Confirmation Process

• What is a suspect or presumptive result?• What is confirmation or follow up?

Hi John,Please note that the below sample is suspect for E.Coli:

Lot: 12345 Food sample A, DOM 15/05/2015

The sample is under confirmation and the final result will be updated once complete

Thank you and best regards

Jenny BloggsMicrobiologist

• Have you ever received an email like this?










What is a suspect result?

For a number of microbiological tests the initial test is generic and any positive or SUSPECT result requires more testing.

A suspect may be:- The micro-organism being analysed- Other micro-organisms present that

interfere- Matrix interference

Synonyms: Presumptive










What is confirmation?

Carrying out further steps of a test for identifying a particular type of bacteria.Therefore CONFIRMING its presence.

When the steps have been completed we say that the test has been “Confirmed”.

After confirmation, the result will be finalised.

Synonyms: Follow up



• What tests can require confirmation?– Coagulase Positive Staphylococcus– E.coli and Coliforms– B. cereus

– Salmonella– Listeria– Clostridium perfringens

– Enterobacteriaceae


High to Low Counts

• How can a result be high in one test and then low when retested?

• This can be caused by a number of factors.– The sample may not be completely homogenous.– Lab error– The sample has anti-bacterial properties.– Natural sample variation – e.g. due to the make-up and/or

components of the sample.


High to Low Counts: Sample Not Homogeneous

• The laboratory generally uses 10g of the sample in the required media, e.g. 90g

• Of that 100g, 0.1g to 1.0g is removed for each test

• A reported result of 500 cfu/g, may only be 5 colonies on an agar plate. This is a low number and with repeat testing small variations will affect the result by 100’s of cfu/g

• In general, acceptable variation would be within half a log, e.g.

Result Possible Variation

5,000 (or 5 x 103) ± 500

3,000,000 (or 3 x 106) ± 500,000


High to Low Counts: Anti-Bacterial Properties

• The sample may contain substances that are anti-bacterial

• Some samples have the capacity to kill bacteria, e.g. cinnamon, vinegar, salt and preservatives

• Samples are stored for 3-5 days from initial testing & before a retest generally occurs. Substances in the sample itself may lower the bacterial count e.g water activity, available nutrients & temperature

• Therefore possible high levels upon initial testing have been killed off or had their numbers lowered before the retest.


High to Low Counts: Lab Error

• This is always a possibility and our quality procedures and controls minimise this risk. However it can never be 100% removed.

• If we believe lab error is a possibility we will try to look for a pattern, e.g.• Do all the similar samples have a high count?• Does the sample have a history of high counts?• Do unrelated samples tested concurrently have high counts?

• We also look at other factors, e.g.• Experience of the technician• Environmental monitoring results• Media testing, including daily positive/negative controls


End.

• Thank you very much for your attention

• If you would like more information on ALS Food, please visit our website or contact one of our team

http://www.alsglobal.com/en/Our-Services/Life-Sciences/Food/Food-Capabilties/Australia-Capabilities

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food microbiology - the basics

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